Mechanism of sulforaphane-induced cell cycle arrest and apoptosis in human colon cancer cells.

Sulforaphane (SFN) is a natural micronutrient found in cruciferous vegetables that has been shown to possess antitumoral properties in carcinogen-treated rats. In vitro, SFN regulates phase II enzymes, cell cycle, and apoptosis. In the present study, we investigated the relationship between SFN induction of apoptosis and cell cycle arrest in HT29 human colon carcinoma cells. In previously published data, a significant increase in the G2/M phase of the cell cycle has been observed in SFN-treated cells that was associated with increased cyclin B1 protein levels. In the present study, our results show that SFN induced p21 expression. Moreover, preincubation of HT29 cells with roscovitine, a specific cdc2 kinase inhibitor, blocked the G2/M phase accumulation of HT29 cells treated with SFN and abolished its apoptotic effect (22.2 +/- 4 of floating cells in SFN-treated cells vs. 6.55 +/- 2 in cells treated with both SFN and roscovitine). These results suggest that the cdc2 kinase could be a key target for SFN in the regulation of G2/M block and apoptosis. Moreover, in SFN-treated cells the retinoblastoma tumor suppressor protein (Rb) is highly phosphorylated. Inhibition of the cdc2 kinase by roscovitine did not change the phosphorylation status of Rb in SFN-treated cells, suggesting that this cyclin-dependent kinase may not be involved. In our study, we did not observe any significant change in the proteasomal activity between control and SFN-treated cells. Moreover, inhibition of proteasomal activity through the use of MG132 diminished SFN-induced HT29 cell death, suggesting that the apoptotic effect of SFN requires a functional proteasome-dependent degradation system. In summary, we have elucidated part of the mechanism of action of SFN in the concomitant regulation of intestinal cell growth and death.

Chronic myeloid leukemia associated with B-cell chronic lymphocytic leukemia: evidence of two separate clones as shown by combined cell-sorting and fluorescence in situ hybridisation.

The simultaneous occurrence of Philadelphia positive chronic myeloid leukemia (Ph+ CML) and B-cell chronic lymphocytic leukemia (B-CLL) is a rare event which raises the possibility that the two malignant clones derive from a common, or distinct, malignant stem cells. In this study, we used combined CD19-based cell-sorting and fluorescence in situ hybridisation (FISH) to investigate whether or not the BCR-ABL fusion gene was present in the malignant B-cells of a patient who presented a Ph+ CML/B-CLL association. The CD19+ cells lacked the BCR-ABL rearrangement whereas all CD19-cells exhibited the fusion gene. This result demonstrates that B-cell transformation occurred in a Ph-B-cell subset.

Contribution of apoptosis in the cytotoxicity of the oxaliplatin-irinotecan combination in the HT29 human colon adenocarcinoma cell line.

Interactions between the topoisomerase I inhibitor irinotecan (CPT-11) and the platinum derivative oxaliplatin (L-OHP) were investigated in HT29 colon cancer cell line. Synergism was observed when cells were simultaneously exposed to drugs or when cells were first exposed to CPT-11. Flow cytometric studies showed a G(2)/M accumulation when cells were exposed to the simultaneous and CPT-11-->L-OHP combinations whereas a persistent S phase delay was observed when cells were first exposed to L-OHP. We characterised the cytotoxic effect by assessing the induction of apoptosis. Irinotecan induced substantial DEVDase activity and poly(ADP-ribose) polymerase cleavage while this activity was moderate and delayed after exposure to L-OHP. Combination experiments showed a sequence-dependent onset of apoptosis, the CPT-11-->L-OHP schedule being the earliest and the most effective; on the other hand the apoptotic signaling generated by CPT-11 was partly inhibited in the simultaneous combination and in the L-OHP-->CPT-11 sequence. Cell death studies using a dual staining technique showed a shift from apoptosis to necrosis when combining these drugs at high concentrations. Synergistic interactions observed using CPT-11 before L-OHP may be linked to an early apoptotic signaling while the L-OHP-induced S phase block could account for the observed additive effect in the reverse sequence. An additional phenomenon might work towards synergism for the simultaneous combination.